Hyperactive EGFR is a critical target for cancer therapeutics. EGFR tyrosine kinase inhibitors (TKIs) are initially quite effective in shrinking tumors harboring EGFR mutations. In contrast, TKIs have shown limited efficacy in tumors lacking mutant EGFR, even when there is evidence of high EGFR pathway activation. In basal-like breast cancer (BLBC), EGFR is often amplified or overexpressed. In fact, recent genomics analysis has identified EGFR-driven expression signatures in 90% of BLBC, suggesting that EGFR-hyperactivation plays a critical role in BLBC biology. Importantly, EGFR expression is higher in patients harboring nodal or distant BLBC metastases. It is therefore important that we address this unmet clinical need and determine why EGFR targeted therapies have failed BLBC patients. The identification of novel proteins involved in regulating EGFR during BLBC development may begin to explain why TKIs have limited efficacy in BLBC patients, and will also provide new opportunities for drug development. We originally identified FAM83B in a forward genetic screen for genes that drive HMEC transformation. Our analysis of FAM83B has generated extensive new information about the function of FAM83B in cancer. We have determined that: (1) FAM83B is significantly elevated in many cancers; (2) elevated FAM83B expression drives EGFR and downstream effector activation, resulting in tumorigenicity; (3) elevated FAM83B expression confers resistance EGFR TKIs; (4) inhibition of FAM83B suppresses EGFR and downstream effector activation and inhibits the tumorigenicity of cancer cells in vivo. These data suggest that FAM83B plays a key role in EGFR signaling, and provides compelling evidence that therapeutically targeting FAM83B may serve as an innovative method to suppress EGFR-driven tumors. Towards deciphering the mechanism by which FAM83B increases EGFR activity, we have identified a protein complex comprising FAM83B and EGFR. Functionally, the FAM83B/EGFR complex increases EGFR activity, as well as EGFR- dependent FAM83B phosphorylation. The FAM83B/EGFR complex is critically important for FAM83B-mediated transformation. Thus, we hypothesize that elevated FAM83B expression nucleates key EGFR signaling complexes that drive aberrant, sustained EGFR effector activation. The objectives of the proposed studies are to (1) determine how elevated levels of FAM83B promote hyperactive EGFR signaling; (2) identify FAM83B- dependent changes in EGFR signaling interactions; and (3) determine the therapeutic efficacy of targeting FAM83B. Upon completion of the proposed studies, we will have a greater understanding of how FAM83B impinges on the regulatory cascades that control EGFR activity. Our studies should shed light on why EGFR- TKIs have shown little efficacy in EGFR-driven BLBC, as elevated FAM83B expression maintains high EGFR activity even in the presence of TKIs. The insight gained from our proposed studies will lay the foundation for targeting FAM83B-mediated EGFR hyperactivation, bringing us closer to overcoming the devastating effects of the EGFR-driven cancers, such as the highly aggressive BLBC.